Integrative Properties of Radial Oblique Dendrites in Hippocampal CA1 Pyramidal Neurons  Attila Losonczy, Jeffrey C. Magee  Neuron  Volume 50, Issue 2, Pages 291-307 (April 2006) DOI: 10.1016/j.neuron.2006.03.016 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Two Forms of Dendritic Integration Revealed by Multisite Two-Photon Glutamate Uncaging in Radial Oblique Dendrites of CA1 Pyramidal Neurons (A) Two-photon image stack of a CA1 pyramidal neuron filled with OGB-1 (100 μM). (B) Single scan image showing the seven spines used for two-photon glutamate uncaging. (C) gluEPSPs for the seven different spine locations are shown with the thicker lines indicating the mean response. (D) Arithmetic sum of the individual responses (one to seven spines) using a 0.1 ms interval. (E) Responses produced by uncaging at a progressively increasing number of the spine locations with a 0.1 ms interval (same uncaging sequence as in [B] and [C]). (F) Expanded gluEPSPs from another oblique branch and first temporal derivatives of the same traces (bottom) (input site at 35 μm from trunk). (G) Plot showing the somatically measured peak gluEPSP amplitude versus the peak amplitude of the arithmetic sum of individual spine responses for uncaging locations (black dashed line is linear summation). (H) Plot of peak δV/δt values versus number of input locations. It can be seen from the individual somatic voltage traces and from the input-output curve that higher levels of synchronous input (five to seven spines at 0.1 ms interval) lead to branch spike generation that appears as a fast component in the upstroke of the suprathreshold gluEPSPs (arrowhead labeled “fast” in [F]), and as a slight supralinearity in the input-output curve. Slower rising portion of gluEPSP indicated by arrowhead labeled “slow” in (F). Notice the corresponding sharp increase in the δV/δt plot at threshold. (I–K) Traces and plots from another branch (input site at 80 μm from the trunk) with gluEPSPs evoked at a 2 ms interval. These events show no step increase in δV/δt and an essentially linear summation over the whole range of input levels (1 to 20 spines). Notice that the level of somatic depolarization achieved is nearly four times that of the threshold level for 0.1 ms interval input (red circle; gluEPSPs not shown). (L) Summary plot of peak δV/δt amplitude (mean ± SEM) showing a sharp increase at threshold for the 0.1 ms interval (n = 92) but not 2–5 ms interval recordings (n = 23). (M) Summary plot of mean input-output curves for threshold (thr)-aligned peak gluEPSP amplitudes (mean ± SEM). A supralinear amplification was observed in the presence of a dendritic spike (n = 92 branches with 0.1 ms uncaging interval), while input-output for 2–5 ms uncaging interval (n = 23) showed only linear summation. See Experimental Procedures for threshold alignment procedure. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Temporal Synchrony of Input Pattern Determines Oblique Dendrite Integration (A) gluEPSPs and plots from 0.1 ms interval uncaging (upper traces/filled circles) show that higher input levels (five spines) lead to dendritic spike generation (pointed to by arrows). Uncaging at the same spine cluster with a 1 ms interval (lower traces/filled triangles) also evoked a dendritic spike with a similar somatic amplitude and rate of rise but at a higher threshold level (input-output plot, 4.9 versus 5.6 mV). (B) Slightly smaller unitary uncaging events at the same location resulted in dendritic spike generation at higher input levels for a 0.1 ms interval (six spines, upper traces/filled circles) but did not elicit a dendritic spike for 1 ms intervals (lower traces/open triangles). Note in (B) that seven uncaging locations given at 1 ms intervals produce a depolarization (5.9 mV) that is comparable to the threshold level for 1 ms interval in (A), yet the slower depolarization in (B) fails to evoke a branch spike. (C) Summary plot of peak δV/δt amplitude (mean ± SEM), which shows a sharp increase at threshold for 1 ms intervals when local threshold is crossed within 6 ms (n = 9) but remains linear when >6 ms are required to reach threshold (n = 12). (D) Summary plot presenting mean input-output curves for threshold (thr)-aligned peak gluEPSP amplitudes (mean ± SEM). A supralinear amplification was observed for 1 ms uncaging interval with dendritic spike (n = 9), while input-output curves obtained from 1 ms interval recordings without dendritic spike (n = 12) showed linear summation. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Amount of Input Required for Local Spike Initiation in Oblique Dendrites (A) Image stack of a CA1 pyramidal cell showing the proximal and distal uncaging locations on an oblique branch. (B) Representative somatic voltage traces recorded in response to uncaging at a progressively increasing number of spines at the two locations with a 0.1 ms interval (the color of the traces at threshold correspond to the colored dots in [A]). The initial rising phases of gluEPSPs are shown on an expanded time scale at right. (C) Plot of actual versus expected peak somatic gluEPSP amplitudes for the two locations. (D) Bar graph showing mean ± SEM threshold for dendritic spike initiation at proximal (5–50 μm; n = 33) and distal (60–126 μm; n = 44) locations. Individual input locations were clustered within a <20 μm segment of the branch. (E) Plot of somatically measured threshold as a function of distance of the input location from the branch point. Black circles connected with lines represent two input locations with different distances on a same oblique branch (n = 16 locations in 8 branches), while gray circles denote single uncaging locations on obliques (n = 76 branches; dashed line is exponential fit to the population data). (F) Plot of unitary gluEPSP amplitude versus distance of spine location from the trunk. Connected symbols represent individual experiments where seven to ten spines distributed across 60–80 μm oblique segments were stimulated with uniform laser power (n = 13 branches). Black dashed line is exponential fit to the population amplitude data, while red dashed line is the scaled exponential fit to the somatic threshold (from Figure 3E). (G) The somatically measured 20%–80% rise time of gluEPSP values are plotted as a function of distance of the individual uncaging location from the trunk along the oblique (n = 13 branches). (H) Image stack of a CA1 pyramidal cell (left). Dashed boxed region is expanded at right to show the 20 spines used for uncaging (red circles). (I) gluEPSPs evoked at 0.1 ms interval at progressively more spines (from 2 to 20). (J) Input-output plot for the data showing dendritic spike initiation at the 14th input. (K) gluEPSPs evoked at 0.1 ms interval for a total of seven spines (upper) or 20 spines (lower) at the same input location. (L) Input-output plot for the data showing dendritic spike initiation at either the 6th or 16th input. Notice that threshold values were very similar regardless of the spine numbers (3.1 versus 2.7 mV, respectively; arrows). (M) Population data for input patterns (mean ± SEM) using either seven to ten (n = 92; open circles) or 20 input spines (n = 26; filled circles). Mean threshold values (arrows) are not different between the two populations. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Single Radial Oblique Branches Function as Single Integrative Compartments (A) Representative somatic voltage recordings in response to uncaging at progressively increasing number of locations for distributed (red traces) and clustered (green traces) configuration with a 0.1 ms interval. The lower traces represent the corresponding temporal derivatives. Dashed lines across the temporal derivatives indicate the subthreshold δV/δt levels. (B) Stack image of the apical dendritic region of a CA1 pyramidal neuron showing positions of the seven spines on a radial oblique branch for clustered (green dots) and distributed (red dots) experimental arrangements. The temporal sequence of distributed locations during uncaging with a 0.1 ms interval is indicated by associated numbers. (C) Plot showing measured versus expected gluEPSP amplitudes for the clustered and distributed recordings shown in (B). (D) Plot of somatically recorded threshold for clustered and distributed patterns as a function of distance from the branch point on the main trunk. The horizontal lines denote the positions and lengths of dendritic segments covered by distributed configurations. (E) Bar graphs showing that the mean ± SEM somatic δV/δt (left) and the mean somatic amplitude (right) of dendritic spikes are similar for clustered (“C”; n = 76) and distributed (“D”; n = 15) configurations. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Input Pattern-Dependent Local Ca2+ Transients in Radial Oblique Dendrites (A) (Top) Single scan image of an oblique dendritic segment (corresponds to the dashed boxed region in the stack image of a CA1 pyramidal neuron at bottom left) showing the spatial arrangement of seven spines selected as uncaging locations. The yellow line indicates the position of the line scan (LS) through spines and the dendritic shaft. (Bottom right) Representative 300 Hz line scans for a 0.1 ms uncaging interval. Arrowheads indicate the time of uncaging. Regions over the dendritic shaft (“D”) were further analyzed offline and presented as ΔF/F in percentage values (ΔF/F traces typically represent averages of two to five trials). (B) Representative uncaging-evoked local Ca2+ transients for synchronous (0.1 and 1 ms intervals w/spike) and asynchronous (5 and 1 ms intervals w/o spike) input patterns. (C) Plots of peak somatic δV/δt (left) and percentage ΔF/F (right) versus expected somatic voltage for recordings with 0.1 ms and 5 ms interval in (B), showing that the step in δV/δt coincides with a step increase in the local Ca2+ transient for synchronous input (arrows indicate spike threshold for 0.1 ms uncaging interval). Asynchronous input (5 ms) does not produce a step increase in either the δV/δt or Ca2+ signal; instead the Ca2+ transient plotted versus expected somatic voltage shows an essentially linear relationship. Red dashed lines represent the slope of the peak amplitude of local Ca2+ signals evoked by subthreshold gluEPSPs (0.1 ms). (D) Summary plot of the threshold-aligned peak values of Ca2+ transients (mean ± SEM) versus relative number of inputs for the four different uncaging conditions (n = 57 for 0.1 ms; n = 9 for 1 ms w/d-spike; n = 8 for 1 ms w/o-spike; and n = 16 for 5 ms). (E) Stack image of a part of the apical dendritic arborization of a CA1 pyramidal neuron showing three different uncaging locations where synchronous (0.1 ms) input patterns were delivered. (F) Appropriately colored traces show threshold Ca2+ signals recorded at like-colored locations. Just subthreshold traces are in gray. (G) Plot of threshold Ca2+ signal versus location showing the uniformity of the spike-associated Ca2+ signal along the branches. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Ca2+ Signals from Dendritic Spikes near the Input Site (A) Single scan image of an oblique branch showing the positions of the uncaging locations (orange dots) and line scans both within (orange line) and outside but near (green line) the input site. Ca2+ transients obtained from the two line scan positions with synchronous (0.1 ms; left) and asynchronous (5 ms; right) uncaging intervals, showing that only dendritic spike initiation evokes substantial Ca2+ signals outside the input location. Also, Ca2+ signals obtained outside the input site (green traces) did not show as additional suprathreshold amounts of input are delivered to the branch. (B) Grouped data of peak ΔF/F values (mean ± SEM) obtained within and in close proximity to the input site plotted versus the relative number of inputs. This plot shows that the two regions experience comparable supralinear step increases at threshold, while signals outside the input location lack any substantial sub- and suprathreshold signals. (C) Ca2+ signals from backpropagating action potentials (bAPs) in obliques are comparable in amplitude with those obtained from local dendritic spikes outside the input site. Position of line scans within (orange) and near (green) the input site (orange dots) in an oblique segment (shown in the middle). Ca2+ signals evoked by synchronous uncaging at a progressively increasing number of spine locations and recorded within and near the input site (left). Single action potentials evoked by brief somatic current injections and their corresponding Ca2+ signals (five consecutive individual traces and the average) recorded at the distal line scan position (right). (D) Summary of the peak Ca2+ transient amplitude (mean ± SEM) evoked by dendritic spikes outside the input site and bAPs. Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Oblique Dendritic Spikes Are Dependent on the Activation of Voltage-Gated Na+ Channels (A) Somatically recorded synchronous gluEPSPs (0.1 ms interval), δV/δt of synchronous gluEPSPs, asynchronous gluEPSPs (5 ms interval), and Ca2+ signals associated with the synchronous gluEPSPs under control condition (top traces; spike pointed by arrow) and in the presence of TTX (1 μM; bottom traces). (B) Input-output plot of the two uncaging intervals in the presence of TTX. (C and D) Summary plot of threshold-aligned (arrow; control threshold was used to align input-output curves in the presence of TTX) peak somatic gluEPSP amplitude (C) and gluEPSP δV/δt (D) versus expected amplitude (mean ± SEM; n = 16) for input given with a 0.1 ms interval. (E) Local Ca2+ transients at the input site in response to a 0.1 ms uncaging in control and in the presence of TTX (mean ± SEM; n = 10). Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 8 Role of NMDARs in Radial Oblique Dendritic Integration and Local Ca2+ Signals (A) Somatically recorded synchronous gluEPSPs (0.1 ms interval; spike pointed by arrow), δV/δt of synchronous gluEPSPs, and asynchronous gluEPSPs (5 ms interval) under control condition (top) and in the presence of NMDAR blocker (50 μM APV; bottom). (B) Input-output plot of the two uncaging intervals in the presence of NMDAR blocker. (C and D) Summary plot of threshold-aligned (arrows) peak somatic gluEPSP amplitude (C) and gluEPSP δV/δt (D) versus expected amplitude (mean ± SEM; n = 32) for input with 0.1 ms interval in control and in the presence of NMDA blockers (pooled data for 50 μM APV and 15 μM MK-801; n = 32). (E) Representative local Ca2+ transients recorded in response to 0.1 ms (left) and 5 ms (right) interval uncaging for control (top) and in NMDAR blocker (bottom) showing that bath application of APV (50 μM) led to a substantially reduced local Ca2+ influx for asynchronous input (5 ms interval) while spike-associated Ca2+ influx is still present during synchronous input. (F) Summary plot of the threshold-aligned peak values of the Ca2+ responses for 0.1 ms uncaging interval (mean ± SEM; n = 22). (G) Summary plot of peak values of the Ca2+ responses for 5 ms uncaging interval versus expected EPSP amplitude (mean ± SEM; n = 11). Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 9 Effects of VGCC Blockers on Input Summation and Associated Local Ca2+ Signals in Radial Oblique Dendrites (A) Somatically recorded synchronous gluEPSPs (0.1 ms interval; spike pointed by arrow), δV/δt of synchronous gluEPSPs, asynchronous gluEPSPs (5 ms interval), and Ca2+ signals associated with the synchronous gluEPSPs in the presence of voltage-gated Ca2+ channel blockers (0.5 μM SNX-482, 1 μM ω-conotoxin MVIIC, and 20 μM nimodipine). (B) Input-output plot for measured versus expected gluEPSP amplitude for a 0.1 ms interval and for a 5 ms interval uncaging in the presence of VGCC blockers. (C) Summary plot of gluEPSP amplitude for 0.1 ms interval uncaging aligned to threshold (mean ± SEM; n = 15; arrows) showing the limited effect of VGCC blockers on spike-associated nonlinearity. (D) Plot of peak temporal derivatives of gluEPSPs (mean ± SEM) for 0.1 ms interval versus expected amplitude showing that the magnitude of the step-like increase at threshold was only slightly reduced by VGCC blockers. (E) Summary data of ΔF/F (mean ± SEM) for 0.1 ms interval versus expected peak somatic voltage responses aligned to threshold (n = 15). Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 10 Effects of A-Type K+ Channel Blockade on Oblique Dendritic Integration and on Associated Local Ca2+ Signals (A) Somatically recorded synchronous gluEPSPs (0.1 ms interval; spike pointed by arrow), δV/δt of synchronous gluEPSPs, asynchronous gluEPSPs (5 ms interval), and dendritic spike-associated Ca2+ signals under control condition (top traces) and in the presence of Ba2+ (150 μM; bottom traces). (B) Input-output plot of the two uncaging intervals in the presence of Ba2+. (C) Plot of grouped data for gluEPSP measured versus expected area demonstrating the prominent role of A-type K+ channels in regulating gluEPSP duration (mean ± SEM; n = 21). (D) Grouped data obtained for 0.1 ms interval gluEPSPs aligned to threshold showing that Ba2+ application did not affect the threshold for the generation of dendritic spike (arrows) but slightly enhanced summation supralinearity at suprathreshold input levels (mean ± SEM; n = 21). (E) Plot of peak temporal derivatives of gluEPSPs for a 0.1 ms interval versus expected somatic voltage (mean ± SEM; n = 21). (F) Summary plot showing the effect of the different blockers on both the spike-associated Ca2+ signal and the linear slope of the subthreshold Ca2+ signal plotted as relative to control (mean ± SEM; control: n = 57; TTX: n = 10; VGCC blockers: n = 15; APV/MK-801: n = 22; Ba2+: n = 34). (G) Grouped data obtained for 5 ms interval gluEPSPs aligned to threshold showing that Ba2+ application enhanced summation such that it became supralinear (mean ± SEM; n = 11). (H) Grouped data of Ca2+ signals associated with 5 ms interval input plotted versus expected amplitude aligned to threshold showing that Ba2+ application increased the amplitude of these signals (mean ± SEM; n = 11). Neuron 2006 50, 291-307DOI: (10.1016/j.neuron.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions